Lactic acid is a commodity biochemical sold in bulk and as a series of specialty derivatives into a competitive market highly sensitive to production costs. Currently, lactic acid is manufactured by synthetic and fermentative methods for use in food manufacturing, pharmaceuticals, leather tanning, metal pickling, and as a starting material in specialized chemical processes. Fermentations involving the production of lactic acid usually involve the conversion of monosaccharides such as glucose, fructose, galactose, or disaccharides such as sucrose or lactose into lactic acid. Certain homofermentative strains producing only lactic acid as a product have been used, such as Lactobacillus delbrueckii, L. casei, L. acideophilus, and L. bulgaricus. L. delbrueckii consumes either sucrose, glucose or fructose, but does not consume lactose. The other three species consume lactose and galactose in addition to the other sugars. However, to improve the efficiency and cost of lactic acid production by fermentative methods, there is a need to find low-cost, high-nutrient media, and to develop cheaper more efficient production systems.
Heat-stable, food-grade lactic acid requires a high degree of purity. Thus, it is not obvious that the production of lactic acid by fermentation of a low-cost, high-nutrient media will result in an economical and simple process for the production of heat-stable lactic acid.
The present invention provides a combination of a continuous fermentation technology utilizing an industrial waste byproduct stream (e.g, cheese whey permeate) as the raw material and of a solvent extraction recovery process allows for the production of lactic acid at a cost below those associated with conventional manufacturing methods, while meeting quality standards. The process is applicable to the production of L(+), D(-), or L(+)D(-) racemic lactic acid. Thus, the term lactic acid will refer to all three of the cited forms.
Continuous high productivity bioreactors may be used to reduce the cost of fermentation-based processes. One such bioreactor is the retained-cell bioreactor (i.e., cell-recycle fermenter) which is a special example of a chemostat (CSTR) fermenter in which increased volumetric productivities are obtainable by continuously separating biomass from the product stream and recycling the biomass to the fermenter. Cross-flow microfiltration or ultrafiltration modules or centrifugation may be used to effect this separation. However, the economic advantage of any continuous process is highly dependent upon a long operating time relative to the batch process. Thus, for the successful operation of a retained-cell bioreactor based on cross-flow filtration, it is critical to minimize such problems as the fouling associated with solutes, especially proteins, which are found in the preferred raw material, such as cheese whey permeate.
Cheese whey permeate, which contains about 5% lactose, is an excellent raw material for lactic acid production because it is a domestic sugar source whose price is competitive with current world sugar prices. The large volumes of whey currently discarded around the U.S. are ample to support the total world lactic acid market volume. Whey proteins are not useful for this invention but may be recovered for sale as high value whey protein concentrate prior to fermentation of the whey permeate to lactic acid. Many cheese manufacturers recover the whey protein concentrate using ultrafiltration, a commercially available technique for removing large molecular weight proteins (greater than 10,000 daltons). However, in practice, this unit operation only recovers about 80% of the whey proteins, with the remainder passing into the whey permeate stream.
Sweet cheese whey permeate is preferred as a raw material. However, the presence of these residual proteins in the whey permeate makes its use as a fermentation feedstock problematical. The residual milk proteins present in cheese whey precipitate upon heating and upon contact with high salt concentrations or organic solvents. This can result in fouled heat transfer surfaces and mass transfer equipment, making the necessary sterilization and recovery operations difficult and expensive. Also, thermal sterilization of whey produces condensation products of proteins which inhibit bacterial growth and lactic acid productivity. Furthermore, the ability of some lactic acid-producing microorganisms to hydrolyze large proteins and peptides is limited which means the proteins in cheese whey may contribute little nutrient value for cell growth. Thus, additional nutrients must be added to a whey-based fermentation medium which adds cost to the final product.
Therefore, it would be desirable to filter sterilize the whey permeate. However, surprisingly, it was found that the proteins remaining in the whey permeate also fouled the sterile filters to an extent that impeded filtration.
This problem of precipitable proteins is particularly relevant to the continuous fermentation and recovery technology described herein. To overcome this problem, the present invention provides a protease treatment for whey permeate that improves upon current technology by allowing longer term operation of the cell-recycle fermenter since it minimizes membrane fouling due to proteinaceous material. A second benefit of this treatment is that the proteins are proteolyzed to amino acids that can subsequently be used as nutrients by the lactic acid-producing bacteria. A third benefit is that amino acids, as opposed to non-proteolyzed proteins, will not interfere with the recovery procedure described herein.
As cited above, the principle source of sugar in whey permeate is lactose at a concentration of about 5%. The volumetric productivity of a batch fermentation using whey permeate as a source of sugar is very low since there is only a short period of fermentation at a high cell concentration before all the lactose is utilized.
Raw materials other than whey permeate may be utilized as starting materials in the process according to the present invention. Any fermentation medium containing at least one of a variety of carbohydrates may be used, such as, glucose, sucrose (such as that in molasses), fructose, maltose, cellobiose, galactose and xylose. Fermentation media containing at least one of these carbohydrates typically also contain one or more of the following: yeast extract, protein hydrolysate (such as, peptones or tryptones), corn steep liquor, and salts of calcium, magnesium, potassium and phosphate.
The concentration of each component of a suitable fermentation medium for lactic acid production is determined by methods familiar to those skilled in the art. For instance, a suitable fermentation medium could contain 50 grams sucrose per liter, 10 grams Bacto-tryptone per liter, 1 gram monobasic potassium phosphate per liter, 0.5 grams magnesium sulfate per liter, 0.1 grams calcium chloride per liter, and 1 gram yeast extract per liter. Any carbohydrate from the above list could be effectively substituted for sucrose in this medium.
If using a fermentation medium as a starting material, other than lactose-containing whey permeate, in accordance with the invention, it will be recognized by those skilled in the art that a protein hydrolysis pretreatment is not essential. Thus, in cases of such starting materials, the fermentation medium may usually be directly subjected to the fermentation microorganism. In a cell-recycle fermenter (e.g, U.S. Pat. No. 3,472,765), the cells are continuously separated from the product stream and returned to the fermenter. Thus, cells that have been grown are not discarded after a single fermentation but are reused. Indeed, it is possible to obtain cell concentrations in the fermenter many times greater than those obtainable in batch fermentation. Therefore, there are several advantages to using a cell-recycle fermenter over a batch fermenter for the fermentation of whey permeate: (1) The long lead times associated with growing the cells from an initial low concentration is not required once steady state has been reached. (2) Much higher cell concentrations (up to 100 grams per liter dry weight) can be reached than those obtainable using batch fermentation. This translates into a greater rate of lactic acid production per unit volume of fermenter. (3) The fermentation is run continuously, allowing product to be pumped downstream to the continuous purification operations without the need for holding tanks. In addition, the fermentation can readily be automated to give a less variable process stream, and there is no down time associated with cleaning and sterilizing the fermenter.
As one aspect of this invention, the cell-recycle fermenter provides other advantages specific to the process for the conversion of whey permeate to heat-stable, food-grade lactic acid: Through suitable choice of fermentation conditions, it is possible to reduce the level of lactose in the product stream to less than 0.01%, while still maintaining economically attractive volumetric productivities. In addition, the fermentation provides this level of performance despite variations of greater than 10% in the concentration of lactose in the waste whey permeate stream. These characteristics are important to the overall process as it is necessary to reduce the residual level of lactose in the lactic acid product stream to as low a level as possible so as to minimize color formation during heating, a principle definition of heat-stable, food-grade lactic acid.
In order to obtain heat-stable lactic acid from the cell-recycle fermentation broth, a simple and inexpensive recovery process is required that selects for lactic acid and against residual carbohydrates, peptides, and color bodies. Such a process, centered on solvent extraction technology, is described herein.
Previous patents (e.g, U.S. Pat. Nos. 3,944,606, 4,275,234, 4,334,095) have disclosed methods for recovering organic acids from aqueous solutions comprising an extraction step whereby the aqueous solution is contacted with a water-immiscible organic solvent containing a secondary, or tertiary amine having at least 20 carbon atoms; the subsequent back-extraction step uses either a temperature or pH differential as the driving force. Forward extraction processes have previously shown that the coefficient of distribution for lactic acid in the water-immiscible extraction liquid is a linear function of increasing the concentration of amine in the organic solvent.
Surprisingly, the present invention provides a method whereby increasing the concentration of amine in the solvent beyond a certain point does not result in a linear increase in the coefficient of distribution of lactic acid: A solution composed of 100% amine does not give the best distribution coefficient for lactic acid. That is, addition of the proper amount of solvent gives a pronounced synergistic effect, depending on the combination of amine and solvent utilized.
The choice of solvent is highly relevant to another feature of this process. Specifically, the invention provides for a high degree of separation between lactic acid and residual sugars during the solvent extraction procedure. The solvents provided are highly selective to lactic acid and reject all the residual sugars in the raffinate. Complete removal of sugars by this process results in heat-stable lactic acid.
Another important aspect of this invention is the preparation of the fermentation broth for extraction. Previous methods of acidification have involved the addition of mineral acids such as sulfuric or hydrochloric. Addition of mineral acids or salts, however, is undesirable since anions from the acid and salts are coextracted with the desired lactic acid.
Thus, an advantage of the invention is that acidification of the fermentation broth may be accomplished by passing the aqueous solution through an appropriate cation exchange resin in the hydrogenated form to achieve the desired pH range. This methodology is particularly necessary in conjunction with the extraction technology of this invention, since acidification by ion exchange has the advantages of removing undesired cations and some color bodies from the broth and of eliminating the addition of anions, all of which might be coextracted with the lactic acid during the solvent extraction step. Thus, acidification by ion exchange will lead to a less complicated and less expensive recovery procedure that will be more readily convertible into a continuous process.
It is the object of the invention to demonstrate a cost-effective fermentation-based process for the production of heat-stable (i.e., food-grade) lactic acid.
To this end, it is an object of the present invention to provide a method for preparing cheese whey permeate for use as a low cost fermentation medium for lactic acid production.
Another object of the present invention is to provide processed whey permeate which may be subjected to filter sterilization, heat sterilization, solvent extraction, or other refining processes without precipitation of proteins.
It is a further object to provide a protease treatment for whey permeate which improves long term operation of a filtration-based cell-recycle fermentation by minimizing fouling of the filtration membrane due to proteinaceous material.
A further object is to provide proteins which are proteolyzed to amino acids that may be subsequently used as nutrients by lactic acid-producing bacteria.
Another object is to provide a method whereby amino acids, as opposed to non-proteolyzed proteins, will not interfere with the recovery procedure.
A further object of the present invention is to utilize a continuous cell-recycle fermentation to obtain high cell densities, and, consequently, a high productivity of lactic acid over an extended period of time.
Another object of the present invention is to obtain a high conversion (approaching 100% of theoretical) of the lactose in whey permeate to lactic acid.
Another object of the present invention is to acidify the resultant fermentation broth without the addition of anions.
Another object of the present invention is to provide an improved method for recovering, purifying, and concentrating lactic acid from whole, centrifuged, or ultrafiltered fermentation broths containing lactic acid.
Another object of the present invention is to use mixed solvents to obtain solvent compositions that have high capacity for lactic acid.
It is a further object of the present invention to provide a method of recovering, purifying and concentrating lactic acid from fermentation broths without the use of temperature gradients.
These and other objects of the invention will be apparent from the following description of the preferred embodiments.